Efficacy In Xenograft Models Research Articles

Abstract 2513: Evaluation of CD37 expression by calibrated IHC identifies non-Hodgkin's lymphoma as a therapeutic target for IMGN529, an anti-CD37-maytansinoid conjugate

Abstract Background: CD37 is a B-cell surface antigen reported to be widely expressed on malignant B cells in patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Expression of CD37 in normal tissues is mainly restricted to B cells in blood and lymphoid tissues. This suggests that CD37 may be an attractive target for development of a therapeutic antibody drug conjugate (ADC). The antibody maytansinoid conjugate, IMGN529 consists of a CD37-binding monoclonal antibody, K7153A, with the DM1 maytansinoid cytotoxic agent attached via an SMCC linker. IMGN529 has demonstrated potent in vitro and in vivo anti-tumor activities supporting its development as a treatment for CD37-expressing lymphomas and CLL. Objective: The objectives of this study were to assess the potential utility of IMGN529 for treatment of lymphoma patients by: (1) determining the extent of CD37 expression in NHL tissue samples relative to CD20, a well-validated target for antibody-directed therapies against B-cell malignancies; (2) identifying xenograft models with similar CD37 expression as seen in NHL patient samples; and (3) evaluating IMGN529 efficacy in these xenograft models. Methods: CD37 expression in human lymphoma cell lines was determined by quantitative flow cytometry. Cell pellet controls were prepared from human lymphoma cell lines that represented a range of CD37 expression to establish a calibrated IHC method. Using this IHC method, CD37 and CD20 expression levels were evaluated on a panel of FFPE biopsy samples from patients with NHL and from NHL-derived tumor xenograft models. For all stained biopsy samples, the extent and intensity of immunostaining was evaluated and antigen expression levels were estimated by comparing to the quantified cell pellet controls. In vivo efficacy was evaluated in subcutaneous xenograft models in SCID mice. Results: Biopsy samples from patients with B-cell NHL including subtypes such as follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and mantle cell lymphoma showed similar prevalence for CD37 and CD20. Most biopsy samples from patients with NHL showed strong and uniform expression levels of the CD37 antigen. As expected, no positive CD37 staining was seen in multiple myeloma and T-cell lymphoma biopsies. IMGN529 was highly active at a single dose of 10 mg/kg in tumor xenografts with CD37 expression levels similar to NHL patient samples. Conclusion: CD37 is expressed widely in B-cell lymphomas, including all major subtypes, which confirms CD37 as target for development of a therapeutic antibody drug conjugate. The CD37-targeting ADC, IMGN529, shows strong in vivo efficacy in lymphoma xenograft models with comparable CD37 expression levels as seen in lymphoma patients. Together these data support the development of IMGN529 for treatment of NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2513. doi:1538-7445.AM2012-2513

Abstract 858: Potent in vivo activity of the aurora kinase inhibitor ABT-348 in human acute myeloid leukemia and myelodysplastic syndrome xenograft models

Abstract The Aurora kinases are a family of serine/threonine kinases that mediate essential functions in cell division. Aurora A depletion results in accumulation of cells in the G2/M phase and apoptosis. Inhibition of Aurora B/C results in abnormal cell division, polyploidy, resulting in apoptosis, therefore, Aurora kinases present an attractive target for chemotherapy. Cells treated with aurora inhibitors enter mitosis with normal kinetics but fail to undergo cytokinesis due to mitotic spindle checkpoint disruption. ABT-348 is a novel adenosine triphosphate (ATP)-competitive inhibitor of Aurora A, Aurora B, and Aurora C (Enzyme IC50 A=116, B=5, C= 1 nM) and a potent inhibitor of all members of the VEGF and PDGF family of receptor tyrosine kinases (RTKs). Despite significant advances in the epidemiological, genetic and biological understanding of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), the basic therapeutic approach has not substantially changed for the last 10-15 years, so most patients still die of this disease. Here we demonstrate profound in vivo efficacy (with regressions) of ABT-348 in both AML (MV-4-11, FLT3 mutant expressing the internal tandem duplication with constitutive kinase activation) and MDS (SKM-1) xenograft models. MV-4-11 tumor-bearing SCID mice were treated at 6.25, 12.5 and 25 mg/kg/day, p.o., q7d x 3 (%TGI ratios on day 30 were 80, 86 and 94%, respectively). SKM-1 tumor-bearing SCID mice were treated at 6.25, 12.5 and 25 mg/kg/day, p.o., q7d x 3 (% tumor growth inhibition or TGI ratios on day 30 were 38, 59 and 80%, respectively). In addition, ABT-348 provided additive effects when combined with cytarabine, decitabine or doxorubicin compared to cytotoxic monotherapies. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of ABT-348 combination strategies in the clinic. Currently ABT-348 is being evaluated (monotherapy and in combination with cytotoxic therapies) in the HL-60 acute promyelocytic leukemia xenograft model in vivo. Dose/scheduling studies for combination therapies in the SKM-1 and HL-60 xenografts are ongoing. Pharmacokinetic/pharmacodynamic biomarker analyses in these xenograft models were evaluated using phospho-H3 (an Aurora B substrate, proliferation) and cleaved caspase-3 (apoptosis) by IHC at various timepoints post single dose (1/2 hr to 5 days). A general decrease in proliferation and increase in apoptosis consistent with the mechanism of action was observed which coincided with the potent in vivo efficacy in xenograft models. Overall, ABT-348 is a potent, oral Aurora kinase inhibitor, demonstrating robust in antitumor activity in AML and MDS xenograft models with a good safety profile that warrants investigation in the clinic. ABT-348 is currently undergoing Phase I clinical trials in advanced hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 858. doi:1538-7445.AM2012-858

Abstract 1386: Predictive markers of efficacy for an angiopoietin-2 targeting therapeutic in xenograft models

Abstract The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and in this era of personalized medicine biomarkers that can predict effect are sorely needed. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. The resulting therapeutic has shown promising activity in a phase 1 clinical trial, with several phase 2 clinical programs in progress/planning. In order to optimize the use of this compound in the clinic a set of predictive biomarkers are desired. Here we describe the construction of a predictive model based on the efficacy of CVX-060 in 13 conventional xenograft models, focusing on renal and ovarian cancer. Pretreatment size tumors from each of the models were profiled for the levels of ∼25 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for ∼12 genes implicated in tumor growth and/or vascularization. The markers chosen for evaluation were accumulated from angiogenesis literature and a differential gene expression analysis of CVX-060 treatment-induced changes. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested via prospective analysis in 7 additional models The results of this work reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-49% TGI, median = 38%). Notably, we define a subset of 5-7 proteins, the levels of which are predictive of TGI by Ang2 blockade. The majority of these proteins are proximal to the target of CVX-060 and the direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. The SNPs and somatic mutations examined here did not correlate with TGI. Our study describes a systematic approach to the problem of identifying predictive markers of anti-angiogenic affect, and demonstrates the significant ground work necessary to reveal treatment-sensitive predictive markers which can be quantitatively defined. This effort has revealed a set of predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1386. doi:1538-7445.AM2012-1386

Abstract B231: Potent in vivo activity of the Aurora kinase inhibitor ABT-348 in a broad spectrum of histological types: Evaluation of efficacy, dosing/scheduling, and PK/PD.

Abstract The Aurora kinases are a family of serine/threonine kinases that mediate multiple essential functions in cell division. Aurora A depletion results in accumulation of cells in the G2/M phase and apoptosis. Inhibition of Aurora B/C results in abnormal cell division, polyploidy, followed by apoptosis, therefore, Aurora kinases present an attractive target for chemotherapy. Cells treated with aurora inhibitors enter mitosis with normal kinetics but fail to undergo cytokinesis due to a disruption of the mitotic spindle checkpoint. ABT-348 is a novel adenosine triphosphate (ATP)-competitive inhibitor of Aurora A, Aurora B, and Aurora C (Enzyme IC50 A=116, B=5, C= 1 nM) and a potent inhibitor of all members of the VEGF and PDGF family of receptor tyrosine kinases (RTK). Here we demonstrate profound in vivo efficacy (with regressions) in a broad spectrum of histological types (breast, colon, NSCLC, HNSCC, melanoma, ovarian, pancreatic, prostate, leukemia, lymphoma and renal; 50–94% TGI, tumor growth inhibition). The treatment was well tolerated with no animal health concerns observed. In addition, ABT-348 was also efficacious in tumor xenografts overexpressing P-gp and provided significant in vivo efficacy in comparison to competitor compounds in colon, NSCLC and ovarian xenograft models. Various dosing schemes to achieve optimal efficacy (IV, OMP and PO) were investigated. The anti-tumor efficacy of ABT-348 was not affected by the route of administration. Similar efficacy was achieved by IV, OMP or PO administration, once weekly in a dose-dependent manner. Pharmacokinetic/pharmacodynamic biomarker analysis in select tumor xenograft models were evaluated by phospho-H3 (an Aurora B substrate, proliferation marker) and cleaved caspase-3 (apoptosis marker) by IHC at various timepoints post single dose (6 hr to 9 days). Significant levels of ABT-348 were detected in the plasma and tumor. A general decrease in proliferation and increase in apoptosis consistent with the mechanism of action was observed and coincide with the potent in vivo efficacy in xenograft models. Overall, ABT-348 is a potent, oral Aurora kinase inhibitor, demonstrating robust in vitro and in vivo antitumor activity with a good safety profile that warrants investigation in the clinic. ABT-348, is currently undergoing Phase I clinical trials in both solid and hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B231.

Abstract B179: Mechanism of antitumor activity of BET inhibitors in models of lymphomas and leukemia.

Abstract Proteins of the BET family bind to acetylated histones and promote gene expression by recruiting transcriptional activators. We developed BET inhibitors and demonstrated a strong association between biochemical inhibition of BET-chromatin binding, MYC suppression and reduced proliferation in hematopoietic cancer cells. We assessed the gene loci affected by BET inhibition by integrating ChipSeq and gene expression profiling, and identified genes that are directly regulated by BRD4 binding, most notably MYC, or genes that are targets of MYC. BET inhibition resulted in robust suppression of MYC expression in tumor xenograft models. Moreover, based on the PK/PD relationships established for these compounds we designed dosing regimens that resulted in significant anti-tumor efficacy in xenograft models of Burkitt's lymphoma and acute leukemia. Compound doses and schedules resulting in sustained MYC suppression were most effective in inhibiting tumor growth and were well tolerated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B179.

Abstract SY20-03: Micelle-based targeting radio-nanotheranostic agents against cancer

Abstract We have developed a novel amphiphilic polymer (telodendrimer), comprised of linear polyethylene glycol and cholic acid cluster, that can self-assemble under aqueous condition to form stable micelles. We have succeeded in loading the nanoparticles with several different hydrophobic drugs. These include paclitaxel, etoposide, vincristine, doxorubicin, bortezomib, sorafenib, lapatinib, temsirolimus, and dexamethasone. This nanoplatform is highly versatile. Particle size can be readily tuned by varying the configuration and composition of the telodendrimer. Nanoparticles of various sizes loaded with DiD, a near infrared hydrophobic dye, were prepared and their biodistribution evaluated in nude mice bearing ovarian cancer xenografts. Particles smaller than 60 nm preferentially targeted the tumors whereas particles at 150 nm were found to concentrate at the liver and lung but with low tumor uptake. The surface chemistry can be easily modified by tethering various acidic or basic peptides to the distal polyethylene glycol tail of the telodendrimers. Undesirable liver uptake was very high for those nanoparticles with high surface charges, either positive or negative. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge was slightly negative. Ligation of tumor targeting ligands to the particle surface further improved tumor targeting and intracellular uptake at the tumor site, resulting in better therapeutic efficacy in xenograft model. In addition to xenograft models, we have also demonstrated that the nanoparticles could preferentially target early spontaneous mammary tumors of PyV-MT transgenic mice. We have just completed a phase I study of placiltaxel-loaded nanoparticles in companion dogs with lymphoma and showed that the drug is safe. Maximum tolerated dose (MTD) was determined to be 97mg/meter square. A phase II study of the same nanoformulation in companion dogs with solid tumor is ongoing. The nanoparticles are stable both in vitro and in vivo. Additional stability of the nanoparticles can be achieved by chemical stitching of adjacent telodendrimers with disulfide bonds after drug loading. Such crosslinked nanoparticles are extremely stable, even in the presence of sodium dodecyl sulfate, a strong ionic detergent. Drug release from crosslinked nanoparticles is much slower than that from non-crosslinked nanoparticles. However, upon addition of 10 mM of glutathione, drug release rate from the crosslinked nanoparticles paralleled that of the non-crosslinked nanoparticles. Since glutathione level is high at the tumor site and inside the tumor cells, it is expected that the disulfide bonds of such crosslinked nanoparticles will be reduced after taken up by tumor cells. Therapeutic studies in xenograft model indeed confirmed that crosslinked paclitaxel-loaded nanoparticle was more efficacious than non-crosslinked nanoparticles. For radioimaging studies, the lysine side chain of telodendrimers was radioiodinated with [I-125]-labeled Bolton-Hunter reagent and then loaded with paclitaxel to form stable [I-125]-labeled nanoparticles. MicroCT/SPECT imaging study with this novel radioprobe in nude mice bearing ovarian cancer xenograft revealed gradual uptake of the radioprobe by the tumor within the first 24 hours. At the 24-hour timepoint, significant amount of radioprobe remained in the circulation. However, by 48 hours, most of the circulating radioprobe disappeared while tumor uptake remained high as late as 94 hours. Liver, spleen, lung and renal uptake were minimal. Together, these studies suggest that an optimal therapeutic nanoparticle should be less than 60 nm in diameter, have slightly negative surface charge, display cancer targeting and pro-endocytotic ligand(s), and be cross-linked by disulfide bonds after drug loading. Such nanoparticle drug is expected to have minimal premature drug release, be preferentially taken up by solid tumors via the enhanced permeability (EPR) effect, enter the tumor cells via the tumor cell targeting ligands, and drug released inside the tumor cells where the glutathione level is high. The addition of an imaging radionuclide to such nanoparticle will make it a highly effective radio-nanotheranostic agent against cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY20-03. doi:10.1158/1538-7445.AM2011-SY20-03

Abstract LB-393: The cancer metabolism inhibitor MPC-9528 induces tumor regression in xenograft models with multiple dosing schedules by causing rapid and sustained reduction in tumor NAD

Abstract Introduction: MPC-9528 is a potent, selective, orally bioavailable inhibitor of nicotinamide phosphoribosyltransferase (Nampt), which catalyzes the rate-limiting step for NAD biosynthesis from nicotinamide. Inhibition of Nampt by MPC-9528 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. MPC-9528 is a potent tumoricidal agent of cancer cell lines of diverse origin and shows anti-tumor activity in multiple xenograft models. Here we explore the determinants of efficacy in xenograft models by comparing the effects of MPC-9528 oral dosing schedules in terms of both tumor NAD depletion and survival. Methods: HT1080 human fibrosarcoma cells were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm3. MPC-9528 was dosed orally, either once weekly, once daily, or twice daily, for a total of two or three weeks. For pharmacodynamic (PD) studies, animals were dosed with MPC-9528 and tumors were collected at various times post-dosing for NAD determination by LC-MS/MS. Results: MPC-9528 (75 mg/kg) dosed once weekly for three weeks in a HT1080 xenograft model resulted in 75% tumor regression on study Day 23, 8 days after the last dose. The ED50 for anti-tumor activity with this schedule was 44 mg/kg and doses at or below 35 mg/kg showed no anti-tumor activity. All doses were equally well-tolerated with <10% change in median body weight. In the same model, MPC-9528 dosed once daily at 10 mg/kg or twice daily at 3 mg/kg for 14 days showed 91% and 92% tumor regression, respectively, by the end of dosing on study Day 15. Two animals in each of the MPC-9528 dose groups showed complete tumor regression. MPC-9528 (4 mg/kg) dosed twice daily resulted in complete tumor regression in 9 out of 10 animals. The plasma concentration versus time profiles of MPC-9528 dosed at 3, 10 or 75 mg/kg indicated that anti-tumor activity was not dependent on maximal concentration of the compound in blood or the total absorbed dose but rather on maintenance of compound concentration above a threshold efficacious concentration. PD studies revealed a rapid and sustained reduction of NAD in tumors from mice dosed with MPC-9528 and that the magnitude and kinetics of NAD change depended on the dose and schedule. Conclusions: MPC-9528 administration results in regression of tumors when plasma concentrations of compound are maintained above a threshold. MPC-9528 shows comparable efficacy when given intermittently, on a weekly schedule, or continuously on a once, or twice daily schedule. This unique attribute of MPC-9528 reflects its mechanism of action as a cell metabolism inhibitor that functions by inhibiting NAD synthesis in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-393. doi:10.1158/1538-7445.AM2011-LB-393

Abstract LB-20: PEGylated TRAIL/Apo2L shows greatly improved circulating half life and improved anti-tumor efficacy in xenograft models for colon and pancreatic cancer

Abstract PURPOSE TRAIL/Apo2L is a death receptor (DR) agonist, inducing apoptosis through the extrinsic pathway in tumor cells expressing DR4 or DR5. Agonistic antibodies or the naked ligand have been tested in the clinic to treat solid tumors. Owing to its extremely short serum half life, TRAIL must be administered by daily IV infusions. Attaching polyethylene glycol (PEG) to the ligand could afford a longer half life and systemic exposure, thus requiring fewer hospital visits for the patient. However, as TRAIL is a functional homotrimer with complex ligand-receptor interactions, non-specific PEGylation will likely result in diminished potency. Using Ambrx's proprietary site specific PEGylation, we evaluated the potency and circulating half life of several PEGylated TRAIL variants. METHODS Using Ambrx proprietary technology, PEG can be attached at a precise location so as not to interfere with receptor binding by engineering insertion of a non-natural amino acid, pAF, which provides a site-specific reactive moiety for attaching other molecules. Several trimeric-PEG-TRAIL analogs were generated and tested for anti-proliferation in vitro and for pharmacokinetic (PK) parameters in the rat. Finally, a PEG-TRAIL analog was tested for anti-tumor activity in pancreatic and colon cancer xenograft model. RESULTS PEGs of varying length (3k, 5k, 10k, and 20k) were attached to site-specifically incorporated pAf to yield one, two or three PEGs per trimeric molecule. One selected PEG analog showed only minimal loss of anti-proliferative activity when tested against a panel of cell lines including representatives from colon, pancreatic, lung and lymphoma cancers. The rat serum half life and AUC of PEG-TRAIL was approximately 6 and 21 fold better as compared to wild type (wt) TRAIL. Administering PEG-TRAIL qdx1 resulted in identical efficacy in pancreatic and colon xenografts as wt TRAIL administered qdx5. Finally, PEG-TRAIL was dramatically more efficacious than Mapatumumab at 1/5 the dose or wt TRAIL at equivalent dose, but had similar efficacy to Lexatumumab. Thus, site specific PEGylation of TRAIL homotrimer produces a drug that maintains in vivo potency and allows intermittent rather than daily dosing. CONCLUSIONS Modifying TRAIL by attaching a single PEG moiety to the homo-trimeric molecule increases rat serum half life compared to wild type TRAIL without decreasing agonistic potency or anti-tumor efficacy in xenograft animal models. There is potential for administering PEGylated TRAIL to patients with pancreatic or colon cancer with a once weekly instead of a daily dosing regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-20. doi:10.1158/1538-7445.AM2011-LB-20

Abstract 3614: In vivo efficacy of the carbonic anhydrase IX (CA9)-targeted antibody-drug conjugate BAY 79-4620 is superior to that of microtubule inhibitors in preclinical models of NSCLC, gastric and colorectal cancer

Abstract BAY 79-4620 is a novel antibody drug conjugate (ADC) consisting of a fully human monoclonal antibody directed against carbonic anhydrase IX (CA9) conjugated to the auristatin derivative monomethyl auristatin E (MMAE) and is currently in Phase I clinical testing. CA9 is overexpressed in a range of tumor types, including gastric cancer, non-small cell lung cancer (NSCLC), pancreatic cancer and colorectal cancer. CA9 is a hypoxia-inducible protein regulated by HIF-1α, and its expression has been linked to higher aggressiveness of tumors and is predictive of poor prognosis in several cancers. In this study, we first compared the internalization rates of the ADC BAY 79-4620 and of the naked antibody, which were found to be similar in several cell lines, indicating that CA9 internalization in vitro is not affected by the conjugated toxophore. It has previously been shown that the cytotoxicity of BAY 79-4620 depends on both CA9 expression and sensitivity of tumor cells to tubulin inhibitors. The anti-tumor efficacy of BAY 79-4620 was therefore compared to that of free toxophore MMAE as well as paclitaxel. At cumulative doses corresponding to a fraction of that of paclitaxel (calculated as toxophore equivalents), BAY 79-4620 showed equivalent or superior efficacy to paclitaxel and MMAE in xenograft models of human NSCLC and gastric carcinoma. Cumulative BAY 79-4620 doses corresponding to only 30% of the highest dose of free MMAE given showed equivalent or superior efficacy in xenograft models of non-small cell lung carcinoma. Moreover, BAY 79-4620 also showed efficacy in patient-derived NSCLC models that are resistant to paclitaxel and/or cisplatin. Finally, regrowing CRC tumors after an initial response to BAY 79-4620 responded well to a second cycle of BAY 79-4620 treatment. This indicates that no resistance through clonal selection of CA9 negative tumor cells was induced and that repeated treatment cycles should be possible for cancer patients. These results show the superiority of the CA9-targeted approach over the systemic administration of tubulin inhibitors, component of treatment regimens commonly used for the treatment of NSCLC and gastric cancer. Efficacy of BAY 79-4620 was particularly high in models expressing high levels of CA9, making BAY 79-4620 a promising agent for the treatment of CA9-positive NSCLC and gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3614. doi:10.1158/1538-7445.AM2011-3614

Paclitaxel Nanocrystals for Overcoming Multidrug Resistance in Cancer

Here we described a paclitaxel (PTX) nanocrystal formulation using d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) as the sole excipient for overcoming multidrug resistance (MDR), a key challenge in current cancer therapy. To the best of our knowledge, it is the first report on PTX nanocrystals which can reverse MDR. TPGS serves as a surfactant to stabilize the nanocrystals and a P-gp inhibitor to reverse MDR. The size and morphology of the nanocrystals were studied by transmission electron microscopy, and the crystalline structure was determined by powder X-ray diffraction. An in vitro drug release profile showed that the nanocrystals exhibited sustained release kinetics compared to Taxol, which is the clinical paclitaxel formulation. The cytotoxicity and antitumor efficacy in xenograft models were also investigated. It is demonstrated that PTX/TPGS nanocrystals have significant advantages over Taxol in achieving better therapeutic effect in Taxol-resistant cancer cells both in vitro and in vivo, which was also confirmed by apoptosis assays. We envision that further development of this type of nanocrystal will provide a novel strategy for drug delivery and multidrug resistance treatment.

Abstract LB-109: Pharmacological evaluation of RP3042, a cytostatic calcium release activated calcium (CRAC) channel inhibitor, in non-small cell lung cancer

Abstract Background: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and is one of the leading causes of cancer-related mortality across the globe. Majority of the NSCLC are characterized by the presence of ras mutation thereby rendering the subject relatively insensitive to treatment by known kinase inhibitors. Current treatment regimes for NSCLC are therefore limited to cytotoxic drugs besides surgery and radiation therapy. Herein, we describe the biological and pharmacokinetic parameters of RP3042, a novel and potent cytostatic agent that inhibits proliferation in NSCLC cell lines via a Calcium Release Activated Calcium (CRAC) channel pathway. Methods: Inhibition of thapsigargin induced calcium influx into Fluo-8 loaded NCI-H460 cells was determined fluorometrically. Viability assay (MTT) was conducted to determine the growth inhibitory effect of the compound on NSCLC cell lines such as A549, NCI-H460, and NCI-H522. Lactate dehydrogenase (LDH) release into the medium was determined colorimetrically. Expression of Orai1 and Stim1 in NSCLC cells was confirmed by RT-PCR. hERG inhibitory potential and selectivity over voltage operated channels (VOC) were estimated by radioligand binding assays. Metabolic stability of the compound was evaluated in microsomes obtained from mouse, rat, dog, monkey, and human. Pharmacokinetic behaviour of RP3042 in plasma after single dose oral administration or IV injection was determined in female Balb/c mice and male Wistar rats. Results: RP3042 demonstrated remarkable potency at inhibiting thapsigargin-induced calcium influx into NCI-H460 cells (IC50 = 72.65 nM). All cell lines tested displayed high expression levels of Orai1 and Stim1, two key proteins responsible for calcium signalling via CRAC channels. Growth inhibition studies indicated that the compound suppressed proliferation in NCI-H460, A549, and NCI-H522 cells with IC50 values of 164, 152, and 206 nM respectively without any noticeable cytotoxicity even at the highest dose. Radioligand binding assays demonstrated the selectivity of RP3042 over VOC with no apparent hERG channel inhibition. Pharmacokinetic studies indicated good oral bioavailability of RP3042 with a maximum plasma concentration of 16.5 and 7.6 µM and an elimination half-life of 2.5 and 1.6 h respectively in mouse and rat. Further, RP3042 was metabolically stable across the species studied besides showing no significant CYP inhibition in human liver microsomes. Conclusions: Results demonstrate the efficacy, safety, and therapeutic potential of RP3042 in NCSLC. Targeting NSCLC through inhibition of CRAC channels represents a novel approach primarily aimed at bypassing the resistance due to ras mutations besides alleviating the deleterious effects associated with cytotoxic agents. RP3042 is currently being tested for its effects on lung cancer biomarkers besides in vivo efficacy in xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-109.

From Natural Products to Small Molecule Ketone Histone Deacetylase Inhibitors: Development of New Class Specific Agents

Histone deacetylases (HDACs) are one of two counteracting enzyme families whose activity controls the acetylation state of lysine protein residues, notably those contained in the N-terminal extensions of the core histones. Deregulation of the acetylation state of specific lysine residues has been implicated in a multitude of biologic processes, notably cancer, where HDACs are known to be involved in the control of cell cycle progression, cell survival and differentiation. HDAC inhibitors are being developed as anti-neoplastic agents. Nature has led the way in the development of these compounds, with trichostatin A being the first hydroxamic acid HDAC inhibitor identified. Likewise, the disulfide depsipeptide Romidepsin is currently in clinical trials, while an array of cyclic tetrapeptides HDAC inhibitors have been reported. Rational drug design has allowed these cyclic tetrapeptide to be transformed into equally potent small molecule inhibitors selective for either class I or class II HDACs. While acyclic alkyl ketones have been demonstrated to be selective HDAC 1, 2 and 3 inhibitors with efficacy in xenograft models, trifluoromethyl ketones have been shown to be selective inhibitors for class II HDACs and recently have been revealed to bind in the active site of the enzyme in their hydrated form.

A Phase I Study of OGX-011, a 2′-Methoxyethyl Phosphorothioate Antisense to Clusterin, in Combination with Docetaxel in Patients with Advanced Cancer

Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2'-methoxyethyl-modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA, has a prolonged tissue half life, enhances drug efficacy in xenograft models, and reduces clusterin expression in humans with a biologically effective dose of 640 mg. The objective of this study was to determine a recommended phase II dose of OGX-011 in combination with docetaxel. Patients with cancers known from the literature to express clusterin were eligible. OGX-011 was given by 2-h i.v. infusion starting at 40 mg weekly after loading doses on days 1, 3, and 5. Docetaxel was given i.v. 30 mg/m(2) weekly for 5 of 6 weeks (schedule A) or 75 mg/m(2) every 3 weeks (schedule B). All patients had serial samples of peripheral blood mononuclear cells and serum assessed for clusterin expression. Forty patients were enrolled to eight cohorts. OGX-011 could be given at the full biologically effective single-agent dose of 640 mg with both docetaxel schedules. Toxic effects were primarily myelosuppression, fatigue, hair loss, gastrointestinal effects (expected docetaxel effects), as well as dose-related chills and fever (expected OGX-011 effects). OGX-011 AUC and C(max) increased proportionally with no apparent effect on docetaxel pharmacokinetics. At the end of cycle 1, serum clusterin showed mean decreases of 34% and 38% (range, 15-99%) at the 640-mg dose levels. OGX-011 can be given at a biologically effective dose with standard doses of docetaxel. Phase II trials of combined OGX-011 and chemotherapy are ongoing in patients with prostate, breast, and lung cancers.

Antitumor Mechanisms of Systemically Administered Epidermal Growth Factor Receptor Antisense Oligonucleotides in Combination with Docetaxel in Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (SCCHN) is one of the most common malignancies worldwide, with low 5-year survival rates. Current strategies that block epidermal growth factor receptor (EGFR) have limited effects when administered as single agents. Targeting EGFR via intratumoral administration of phosphorothioate-modified antisense oligonucleotides has antitumor efficacy in xenograft models of SCCHN. Because intratumoral delivery of therapeutic agents has limited clinical application, the present study was undertaken to examine the therapeutic mechanisms of systemically delivered phosphorothioate-modified EGFR antisense oligonucleotides alone, or in combination with docetaxel, in a SCCHN xenograft model. EGFR antisense oligonucleotides were administered at 5 mg/kg i.p. daily in athymic mice bearing 1483 human SCCHN xenografts alone or in combination with docetaxel at 2.5 mg/kg i.p. once a week for 4 weeks. Administration of EGFR antisense oligonucleotides in combination with docetaxel improved antitumor efficacy and resulted in lower expression levels of EGFR, fewer proliferating cells, and more apoptotic cells in the tumors compared with controls. Systemic administration of phosphorothioated EGFR antisense oligonucleotides for 30 days increased the retention of docetaxel in the tumor by approximately 4-fold compared with tumors treated with docetaxel alone or docetaxel and EGFR sense oligonucleotides (P < 0.05). Combination of EGFR antisense oligonucleotides with low doses of docetaxel has antitumor efficacy, and it may be an effective treatment strategy for SCCHN.

Antibody-Drug Conjugates with Uncleavable Linkers for the Treatment of Non-Hodgkin Lymphoma.

Antibody-drug conjugates (ADCs), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Our research has focused on ADCs that contain the maytansinoid DM1, monomethylauristatin E (MMAE), or monomethylauristatin F (MMAF), all of which are potent inhibitors of microtubule polymerization. These drugs are attached to the antibody via several different linker formats (Table 1).Table 1Linker-drugLinker MechanismLinked viaDrug Cell PermeablePAB-vc-MC-MMAECleavage of peptide bondCysteineYesMC-MMAFUncleavableCysteineNoSPP-DM1Reduction of disulfide bondLysineYesMCC-DM1UncleavableLysineYesWe identified seven cell surface proteins expressed in non-Hodgkin lymphoma (NHL) with limited expression patterns in normal tissue (CD19, CD20, CD21, CD22, CD72, CD79, and CD180), as potential therapeutic targets for ADCs. ADCs directed to any of these seven targets are effective in xenograft models when containing linkers that can be cleaved in the tumor microenvironment or when internalized by tumor cells. However, ADCs with uncleavable linkers, i.e., linkers that are active when antibody is internalized and degraded within cells, are effective only when targeted to CD22 or CD79b. This suggests that these antigens provide the desired intracellular targeting of the cytotoxic drug. Further, we demonstrated that the uncleavable-linker anti-CD22 and anti-CD79b ADCs result in complete tumor regression with no recurrence in some xenograft models of NHL. In vitro experiments revealed that sensitivity of a given cell line to the ADCs anti-CD22-MCC-DM1 and anti-CD79b-MCC-DM1, correlated more with sensitivity of the cell line to the corresponding free drug, than to the amount of surface expression of the target or the amount of internalized ADC. We conducted pilot safety studies in rats (non-binding species) and cynomolgus monkeys (binding species). In rats, ADCs with uncleavable linkers caused less target-independent hepatic and hematologic toxicity than their counter parts with cleavable linkers, likely due to their decreased systemic release of free drug. Because of these results, we evaluated three ADCs with uncleavable linkers (anti-CD22-MCC-DM1, anti-cynomolgus monkey CD79b-MCC-DM1, and anti-CD22-MC-MMAF) at ∼30 mg/kg q3 weeks for 2 doses in cynomolgus monkeys. Normal B-cells were depleted in circulation and in tissue, particularly in germinal centers of lymphoid tissues; an expected pharmacological effect. Other findings included clinically tolerated and reversible elevations in liver enzymes (MCC-DM1 and MC-MMAF conjugates) and decreases in platelet counts (MCC-DM1 conjugates); and minimal to mild sciatic nerve degeneration (MCC-DM1 conjugates). The data demonstrate that anti-CD22 and anti-CD79b ADCs with uncleavable linkers have potent efficacy in xenograft models of NHL, favorable safety profiles in pilot animal studies, and suggest that for specific targets, uncleavable linkers provide a promising mechanism to improve margins of safety in humans.

A Small-Molecule Inhibitor of Bcl-XL Potentiates the Activity of Cytotoxic DrugsIn vitroandIn vivo

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

SGN-35, an Anti-CD30 Antibody-Drug Conjugate, Exhibits Potent Antitumor Activity for the Treatment of CD30+ Malignancies.

Hodgkin/Reed-Sternberg (H-RS) and anaplastic large cell lymphoma (ALCL) cells express high levels of the cell surface receptor CD30, a member of the TNF receptor superfamily. To augment the antitumor activity of anti-CD30 based therapies, a novel synthetic anti-mitotic agent, monomethyl auristatin E (MMAE), has been conjugated to the anti-CD30 antibody cAC10. MMAE was synthetically modified to include a maleimide for conjugation to an antibody and a protease cleavable Val-Cit peptide linker to release the drug from the antibody. The in vitro and in vivo activity of SGN-35, the resulting antibody-drug conjugate, is outlined. SGN-35 has an average of four MMAE molecules per antibody. Cell surface binding, internalization and intracellular release of MMAE were monitored by immunofluoresence and isotope labeling methods. The H-RS cell line L540cy translocates SGN-35 to lysosomes within 16 hours. Analysis of the cellular contents demonstrated that MMAE is rapidly and efficiently liberated from the antibody, and MMAE accumulates inside the cells. After treating L540cy cells with 200 ng/mL of SGN-35 (6 nM MMAE), the intracellular concentration of MMAE reaches 800 nM after 24 hours, rising to nearly 1000 nM after 72 hours. From 24 to 72 hours, about 70% of the intracellular drug is liberated MMAE, while 30% is still bound to the antibody. In vitro potency of SGN-35 was measured by incubating various concentrations of SGN-35 with CD30+ tumor cells and measuring cell viability in culture. SGN-35 had potent, selective in vitro activity with IC50s ranging from 4 to 35 ng/mL for CD30+ cell lines, and an IC50 > 1000 ng/mL for the CD30− line WSU-NHL. This demonstrates that the conjugation of the novel cytotoxic agent MMAE to an antibody can be accomplished without compromising the antibody's specific binding and internalization. SCID mice were implanted with L540cy and treated with intravenous doses of SGN-35. In mice bearing L540cy xenografts, complete tumor regressions were achieved at doses as low as 2 mg/kg q4dx4. SGN-35 is also highly effective in treating mice with disseminated L540cy implants, at well tolerated doses. Based on the potent cytotoxicity, efficacy in xenograft models, and tolerability, SGN-35 is being advanced toward clinical development in CD30-positive hematologic malignancies.

AGS-PSCA: A FULLY HUMAN ANTIBODY TO PSCA FOR THE TREATMENT OF PANCREATIC CANCER

Prostate Stem Cell Antigen (PSCA) is a novel GPI-linked cell surface antigen that is expressed on the majority of primary and metastatic prostate, pancreatic and bladder cancers (Reiter et al., PNAS, 95:1753, 1998; Amara et. al., Cancer Res. 61: 4660, 2001; Argani et. al., Cancer Res. 61: 4320, 2001). Using XenoMouse® technology we selected AGS-PSCA, a fully human IgG1K monoclonal antibody (MAb), as the therapeutic antibody product, based on its high affinity (Kd = 2 × 10−10 M) and efficacy in xenograft models that represent all relevant cancer indications. In our studies with human pancreatic xenografts, grown orthotopically in the mouse pancreas, AGS-PSCA significantly inhibited tumor growth, resulting in substantial increase in the life span of the treated mice. In addition, treatment with AGS-PSCA inhibited tumor spread from the pancreas to distal sites. Enhanced anti-tumor activity of AGS-PSCA was demonstrated when AGS-PSCA was administered with Gemcitabine. AGS-PSCA also significantly inhibited both the orthotopic growth of androgen-independent and -dependent prostate tumors and their metastatic spread. Enhanced anti-tumor activity was demonstrated when AGS-PSCA was administered in combination with Docetaxol. Toxicology and pharmacokinetic preclinical studies in animals indicated that all administered antibody doses were well tolerated and demonstrated a long serum half-life. AGS-PSCA strong efficacy data, together with its pre-clinical safety profile, support the therapeutic potential of AGS-PSCA as a monotherapy and in combination with chemotherapeutic agents for the treatment of pancreatic cancer. Activities are underway to initiate human clinical trials with AGS-PSCA.

A Phase I Pharmacokinetic and Pharmacodynamic Study of OGX-011, a 2′-Methoxyethyl Antisense Oligonucleotide to Clusterin, in Patients With Localized Prostate Cancer

Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA and has been reported to inhibit clusterin expression and enhance drug efficacy in xenograft models. The primary objective of this clinical study was to determine a biologically effective dose of OGX-011 that would inhibit clusterin expression in human cancer. Subjects (n = 25) with localized prostate cancer with high-risk features who were candidates for prostatectomy were treated with OGX-011 by 2-hour intravenous infusion on days 1, 3, and 5 and then weekly from days 8-29 combined with androgen blockade starting on day 1; prostatectomy was performed on days 30-36. Six different doses were tested, from 40 to 640 mg. OGX-011 plasma and prostate tissue concentrations were measured by an enzyme-linked immunosorbent assay method, and the pharmacokinetics of OGX-011 were determined from these data. Prostate cancer tissue, lymph nodes, and serial samples of peripheral blood mononuclear cells were assessed for clusterin expression using quantitative real-time polymerase chain reaction and immunohistochemistry. All statistical tests were two-sided. Only grade 1 and 2 toxicities were observed. The plasma half-life of OGX-011 was approximately 2-3 hours, and the area under the concentration versus time curve and CMAX (peak plasma concentration) increased proportionally with dose (Ptrend < .001). OGX-011 in prostate tissue increased with dose (Ptrend < .001). Dose-dependent decreases in prostate cancer and lymph node clusterin expression were observed by polymerase chain reaction of greater than 90% (Ptrend = .008 and Ptrend < .001, respectively) and by immunohistochemistry (Ptrend < .001 and Ptrend = .01, respectively). OGX-011 is well tolerated and reduces clusterin expression in primary prostate tumors. The optimal biologic dose for OGX-011 at the schedule used is 640 mg.

STA-4783 in combination with paclitaxel induces heat shock protein 70 (hsp70) in a phase I trial

2011 Background: STA-4783 (S) is a bis-thiobenzoylhydrazide that promotes induction of hsp70 and enhances paclitaxel (P) antitumor efficacy in xenograft models. Its mechanism appears to be immunologic, involving stimulation of cytotoxic responses. Methods: A phase 1 trial was conducted in combination with P in advanced solid tumor patients. S and P were coadministered iv over 3 h every 3 weeks. Starting doses were 44 mg/m2 S and 135 mg/m2 P. P was then increased to 175 mg/m2, followed by escalation of S to establish the maximum tolerated dose based upon first cycle toxicity in 3–6 patients at each dose level. Pharmacokinetic (PK) studies were performed during cycle 1 using LC/MS assays to measure both compounds in plasma. Hsp70 was measured in plasma before and after treatment. Results: 35 patients have been evaluated at 8 dose levels. The S dose was escalated from 44 to 525 mg/m2 in combination with P 175 mg/m2. No serious adverse events specifically attributable to S have been observed. P related dose limiting toxicities occurred in a single patient in each of the top three dose levels (neutropenia, arthralgia, and febrile neutropenia with mucositis) resulting in cohort expansion. S exhibited linear PK that was unaffected by P dose. It was rapidly eliminated from plasma with terminal-phase half-life of 0.94 ± 0.23 h and total body clearance of 28 ± 8 l/h/m2. Its apparent volume of distribution was comparable to total body water (Vss, 23 ± 16 l/m2). P PK appeared to be moderately dependent on the S dose, as indicated by a significant trend toward decreasing clearance, an increase in peak plasma concentration and Vss, but without affecting the terminal phase half-life. These observations are consistent with competitive inhibition of P hepatic metabolism. Increased toxicity at higher dose levels was consistent with moderate increase in systemic exposure to P. Induction of hsp70 protein in plasma was dose-dependent peaking at 8 h after dosing. Conclusions: The S/P combination was well tolerated, with adverse events consistent with those expected for P alone. Phase 2 clinical trials are underway in NSCLC, melanoma, and soft-tissue sarcoma. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Synta Synta Synta Synta